Qualifying SPR immunogenicity assayslanding1.gehealthcare.com/rs/005-SHS-767/images/Kuehne SPR Works… · • Biomarker, Pharmacokinetics, Immunogenicity ... Bioagilytix / IPM Biotech

Qualifying SPR immunogenicity assaysDr. Christian Kühne

© 2017 BioAgilytix. 2

Bioagilytix / IPM Biotech

• global CRO for large molecule bioanalysis• supporting pre-clinical & clinical studies• Biomarker, Pharmacokinetics, Immunogenicity



Implemented SPR technology for:

• GLP and GCP-compliant Immunogenicity testing• Drug candidate profiling• GMP batch release testing




• Immunogenicity testing





ADA Screening

binding of ADA´s toimmobilized drug





ADA Confirmatory

differentiation betweenspecific and unspecificbinding by adding excessdrug in mobile phase

ADA Screening






ADA Isotyping

determination of Igsubclass by addingsecondary anti-Igantibody on top

ADA Confirmatory

differentiation betweenspecific and unspecificbinding by adding excessdrug in mobile phase

ADA Screening



Introduction

• Biologic produced with a His-tag for purification• His-tag enzymatically removed after purification

• Immunogenic byproducts/residuals ?

His Hisenzyme


Introduction

• If ADA´s are formed, authorities ask against which target.

HisHis


Qualification

• Biacore T200• CM5 Chip:

Fc1 Fc2 Fc3 Fc4

Blank HisHis


Qualification

• Chip pH Scouting

-500

0

500

1000

1500

2000

2500

3000

0 2 4 6 8 10 12 14

Result Plot

Rel

ativ

e R

espo

nse

- Sto

p

RU

File & Cycle

10 mM Acetate 4

10 mM Acetate 4.5

10 mM Acetate 5

10 mM Acetate 5.5

G-CSF His-G-CSF His

Best results

G-CSF: pH 4.5His-G-CSF: pH 4.5His: pH 4.0


Qualification

• Ligand Immobilization

Fc1 Fc2 Fc3 Fc4

Blank HisHis

74 RU 1960 RU 1900 RU


Qualification

• Sensorgram & Report Points

-1000

-800

-600

-400

-200

0

200

400

600

800

1000

0 100 200 300 400 500 600 700

Sensorgram Example G-CSF Fc4-1RU

Res

pons

e (0

= b

asel

ine)

sTime

base

line

stab

ility

sample regeneration


Qualification

• Chip Regeneration Test – Binding response


Qualification

• Chip Regeneration Test – Baseline response


Qualification

• Chip Stability

40

60

80

100

120

140

160

180

200

220

0 5 10 15 20 25 30

Stability

Rel

ativ

e R

espo

nse

- Sta

bilit

y la

te

RU

Cycle

1

2

3

4


Qualification

• Minimum Required Dilution (MRD)

His 5% serum His-G-CSF 5% serum G-CSF 5% serum

Positive Controls anti-GCSF and anti-His from 10 µg/ml to 1.25 µg/ml in 100 % serum


Qualification





Qualification





Qualification

• Cut Point – G-CSF (FCP = NC.IS + 8.3 RU)

-40

-20

0

20

40

60

80

0 10 20 30 40 50 60 70 80 90

G-CSF

Rel

ativ

e R

espo

nse

- sta

bilit

y

RU

Cycle

Control sample

Sample

30 individual negative patient sera

outlier removal by box plot analysis

Mean of NC (in-study) + CF


Qualification

• Cut Point – G-CSF (FCP = NC.IS + 8.3 RU)

-40

-20

0

20

40

60

80

0 10 20 30 40 50 60 70 80 90

G-CSF

Rel

ativ

e R

espo

nse

- sta

bilit

y

RU

Cycle

Control sample

Sample


Qualification

• Cut Point – His-G-CSF (FCP = NC.IS + 1.6 RU)

-20

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70 80 90

His-G-CSF

Rel

ativ

e R

espo

nse

- sta

bilit

y

RU

Cycle

Control sample

Sample





Qualification

• Cut Point – His-G-CSF (FCP = NC.IS + 1.6 RU)

-20

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70 80 90

His-G-CSF

Rel

ativ

e R

espo

nse

- sta

bilit

y

RU

Cycle

Control sample

Sample


Qualification

• Cut Point – His (FCP = NC.IS + 19.5 RU)




-25

-20

-15

-10

-5

0

5

10

15

20

25

0 10 20 30 40 50 60 70 80 90

His

Rel

ativ

e R

espo

nse

- sta

bilit

y

RU

Cycle

Control sample

Sample


Qualification

• Cut Point – His (FCP = NC.IS + 19.5 RU)

-25

-20

-15

-10

-5

0

5

10

15

20

25

0 10 20 30 40 50 60 70 80 90

His

Rel

ativ

e R

espo

nse

- sta

bilit

y

RU

Cycle

Control sample

Sample


Qualification

• Sensitivity – G-CSF


G-CSF

calculated assay sensitivity [µg/ml]

Ligand Analyte

anti-GCSF anti-His His n.a. 8.301

His-G-CSF 0.120 2.198 G-CSF 0.937 n.d.


Qualification

• Sensitivity – His-G-CSF



Ligand Analyte


His-G-CSF 0.120 2.198 G-CSF 0.937 n.d.

His - G-CSF


Qualification

• Sensitivity - His



Ligand Analyte


His-G-CSF 0.120 2.198 G-CSF 0.937 n.d.

His


Qualification

• Reproducibility - intra assayG-CSF - 5 duplicates in one run

- antibody mix injected- all CV´s below 20%

red line: mean NC + 2x SD-> acceptance criteria set10 µg/ml PC mix to monitor chip stability


Qualification

• Reproducibility - inter assayG-CSF

Positive Control10 µg/ml

Negative Control0 µg/ml

High intra assay sample5 µg/ml

Low intra assay sample1,25 µg/ml red line: mean NC + 2x SD

-> acceptance criteria set10 µg/ml PC mix to monitor chip stability

- 3 independent runs (duplicates each)- merged in one evaluation file- antibody mix injected- all CV´s below 20%


Summary

• Summary

His 19.5 RUHis-G-CSF 1.6 RUG-CSF 8.3 RU

Cut Point CF


Summary

• Summary


Cut Point CFcalculated assay sensitivity [µg/ml]

Ligand Analyte


His-G-CSF 0.120 2.198 G-CSF 0.937 n.d.


Summary

• Summary


Cut Point CFcalculated assay sensitivity [µg/ml]

Ligand Analyte


His-G-CSF 0.120 2.198 G-CSF 0.937 n.d.

His 5.2 %His-G-CSF 3.6 %G-CSF 2.8 %

Reproducibility Intra

CV (highest)

His 4.8 %His-G-CSF 3.4 %G-CSF 3.6 %

Reproducibility Inter

CV (highest)


Sample Analysis

• Sample analysis performed:

comparison between pre-dose and post-dose samplespost-dose had to be 3x higher than pre-dose to be considereddrug induced / treatment emergent


Thank you for your attention

Thank You

Documents

Qualifying SPR immunogenicity assayslanding1.gehealthcare.com/rs/005-SHS-767/images/Kuehne SPR Works… · • Biomarker, Pharmacokinetics, Immunogenicity ... Bioagilytix / IPM Biotech